Method of constructing transgenic bird using lentivirus vector and transgenic bird obtained thereby

ABSTRACT

The present invention provides a lentivirus vector which comprises a coat protein containing VSV-G; a recombinant bird host which is constructed by infecting a bird host with the lentivirus vector; a method of constructing the same; a transgenic chimeric bird which is constructed by infection with the lentivirus vector and offspring thereof. The present invention also provides a method of constructing a G0 transgenic chimeric bird and offspring thereof which comprises infecting fertilized bird egg early embryos with a lentivirus vector comprising a coat protein containing VSV-G, and incubating the embryos; and a G0 transgenic chimeric bird and offspring thereof which is constructed by the method mentioned above. The present invention provides a transgenic bird which is constructed by infecting a bird early embryos with a lentivirus vector comprising a coat protein pseudotyped with VSV-G.

TECHNICAL FIELD

This invention relates to a method of introducing an exogenous gene intoa cultured bird cell using a lentivirus vector. The invention alsorelates to a method of constructing transgenic chimeric birds byintroducing an exogenous gene into fertilized egg early embryos using alentivirus vector and to G0 transgenic chimeric birds obtained by saidmethod.

BACKGROUND ART

In recent years, a number of antibody drugs have been put on the market.However, they are currently produced only by means of animal cellreactors and therefore are expensive. This is a dominant cause ofrestricted spread thereof. The transgenic animal factory technology hasbeen attracted attention as means for cheaply producing proteins havinga complicated structure and requiring a sugar chain for their stabilityand activity manifestation, for example monoclonal antibodies to be usedas drugs.

The attempts to enable mammals such as cows, goats and sheep to producephysiologically active substances cheaply in the milk will soon be putto practical use. In the case of these animals, however, it is adrawback that the period between the construction of transgenics to theinitial production of the desired products is as long as 1 to 2 years.

On the other hand, transgenic birds the sexual maturation period ofwhich is short and which are to produce physiologically activesubstances in eggs thereof are promising animal factories since domesticfouls are efficient protein-producing animals. In constructing stabletransgenics by introducing an exogenous gene into bird somatic cells,however, particular difficulties are encountered. Hence, transgenicbirds have not yet put to practical use (cf. WO 03/014344 A2, ViragenIncorporated (2001)).

One of the reasons why the construction of transgenic birds is difficultis that it is very difficult to obtain fertilized eggs thereof at theone-cell stage and artificially cultivate them. Perry operativelyobtained precleavage chicken cells and established a system forextracorporeally cultivating them (cf. Perry, M. M. (1988), Nature,331). However, it is still impossible to construct transgenics bynucleus transplantation using this technology, since the bird cellpronucleus is covered with yolk and therefore is indistinguishable.

Therefore, the exogenous gene transfer into fertilized bird eggs isrestricted to gene injection into cytoplasm by lipofection orelectroporation, for example. It is impossible to intentionallyintegrate a transgene into a chromosome.

As a result of intensive investigations made by the present inventors,they found out a method of constructing transgenic birds by using areplication-defective retrovirus vector, which is safe and is alsoapplied in gene therapy, for efficient introduction of a desired gene(cf. Japanese Kokai Publication 2002-176880). This has made it possibleto construct transgenic birds carrying a plurality of copies of atransgene in a safe and efficient manner. It was also found that whenthis technique is used, the transgene can be transmitted to the nextgeneration with high efficiency, bringing the utilization of transgenicbirds as substance production systems close to practical use.

The inventors also found out a method of causing protein expressionbased on the novel finding that the inactivation of a gene transferredwith a retrovirus depends on the time of transfer.

The method of gene transfer using a retrovirus vector is currently theonly method of constructing transgenic birds by integrating an exogenousgene into a chromosomal DNA with high transfer efficiency. However, itis a problem that the exogenous gene transferred with a retrovirusvector is inactivated by methylation (the phenomenon called silencing),with the result that the desired protein cannot be expressed.

The silencing phenomenon is considered to be a kind of protective systemof organisms but presents an obstacle in constructing transgenics asanimal factories.

The lentivirus vector developed by Verma et al. is known to be capableof allowing gene transfer even into cells that have differentiated andstopped proliferating, for example nervous system cells withoutaccompaniment of inactivation of the transgene, and it is tolerant ofsilencing (cf. Verma, I. M. et al. (2000) Nat. Rev. Genet. 1, 91).

Pfeifer et al. succeeded in constructing transgenic mice using suchlentivirus vector and reported about the exogenous protein expression insomatic cells thereof (cf. Pfeifer, A. et al. (2002) Proc. Natl. Acad.Sci. USA, 99, 2140).

An attempt to apply this lentivirus vector in constructing transgenicbirds has been reported. Generally, however, bird cells are not infectedwith lentiviruses.

The inventors have made attempts to develop an effective method of genetransfer into birds based on the finding that silencing hardly occurs incertain retrovirus species, side by side investigations concerning thepractical use of transgenic birds through elucidation of the mechanismsof transgene inactivation, as mentioned above. The application ofvectors derived from lentiviruses belonging to the family ofretroviruses is one of such attempts.

Since, however, bird cells are not infected with lentiviruses as such,it is necessary to develop lentivirus vectors capable of infecting birdcells so that lentiviruses may be applied in constructing transgenicbirds.

Lentivirus vectors are a kind of retrovirus vectors, as mentioned above,but they have different properties as compared with common retrovirusvectors, for example vectors derived from Moloney murine leukemia virus(MoMLV).

Generally, retrovirus vectors infect only cells in the growth phase, sothat they cannot serve in gene transfer into cells in the quiescentstate after differentiation. However, lentivirus-derived novel vectorscan infect not only proliferating cells but also quiescent cells, forexample nerve cells and, therefore, have a wide range of application. Itis also known that the transgene inactivation (silencing), which is aproblem with ordinary retroviral vectors, does not occur in them.

There is no report about the infection of bird cells with a lentivirusand, therefore, for applying lentivirus vectors in constructingtransgenic birds, it is necessary to contrive to infect bird cells withthe vector.

SUMMARY OF THE INVENTION

The present inventors made various investigations and found that alentivirus vector comprising a coat protein pseudotyped with VSV-G canallow exogenous gene transfer into cultured chicken cells. Such findinghas now led to completion of the present invention.

Thus, the invention provides, in a first aspect thereof,

a method of constructing a transgenic bird and offspring thereof

which comprises infecting fertilized bird egg early embryos with a VSV-Gpseudotyped lentivirus vector; and,

in a second aspect thereof, the invention provides

a lentivirus vector, a recombinant bird host and a transgenic birdconstructed by said method.

More specifically, the invention provides

a lentivirus vector

which comprises a coat protein containing VSV-G;

a recombinant bird host

which is constructed by infecting a bird host with said lentivirusvector;

a method of constructing said recombinant bird host;

a transgenic chimeric bird

which is constructed by infection with said lentivirus vector, and

offspring thereof;

a method of constructing a G0 transgenic chimeric bird and offspringthereof

which comprises infecting fertilized bird egg early embryos with alentivirus vector comprising a coat protein containing VSV-G, andincubating the embryos; and

a G0 transgenic chimeric bird and offspring thereof

which is constructed by the method mentioned above.

Pfeifer et al. referred to above infected mouse fertilized eggs with alentivirus by treating the fertilized eggs with acidic Tyrode's solutionto thereby removing the clear zone. The VSV-G pseudotyped lentivusescreated by the present inventors can infect untreated bird cells. Theinfectious lentivirus vectors facilitating the pretreatment of cellsconstitute an aspect of the present invention.

One of the advantages of the application of lentivirus vectors inconstructing transgenic birds is that it is highly possible to producefull-body offspring (G1) by gene transfer into the germline. Anotheradvantage is that the transgene-derived protein production increases asa result of the avoidance of silencing.

Thus, the invention discloses a method of constructing transgenic birdsfavorable for protein production. It also discloses transgenic birdsconstructible by such method.

The transgenic birds constructed by the above method can be utilizing inprotein production and, further, in bird breed improvement.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the invention is described in detail.

As the lentivirus vector to be used in accordance with the invention,there may be mentioned HIV (Human immunodeficiency virus)-derived andBIV (Bovine immunodeficiency virus)-derived lentivirus vectors, and thelike. Preferred as the lentivirus vector to be used in the practice ofthe invention are HIV-derived ones. More preferred are HIV-1-derivedones.

From the safety viewpoint, it is desirable that the virus to be used asthe gene transfer vector be replication-defective as a result ofelimination of the regulator genes necessary for replication and, at thesame time, be such that the expression of the virus is effected by lateradding plasmids carrying the genes necessary for virus particlereplication as divided into gene segments.

In accordance with the invention, a virus vector comprising a coatprotein artificially pseudotyped with VSV-G (derived from vesicularstomatitis virus) is used to enable the infection of host birds with thevirus vector. The receptor of VSV-G is a phospholipid, which is amembrane constituent, and this commonly occurs in bird cell membranes aswell and thus makes lentiviruses, which generally cannot infect birdhosts, capable of infecting them.

As the bird hosts, there may be mentioned fertilized bird egg earlyembryos and bird host cells, and the like.

The pseudotyped virus vector prepared by utilizing packaging cells, ahelper virus or the like is introduced into fertilized bird egg earlyembryos and also introduced into bird host cells via intravascular andintracardiac routes preferably by the conventional microinjectiontechnique (Bosselman, R. A. et al. (1998) Science 243, 533). Conceivableas other methods of gene transfer are lipofection and electroporation,and the like.

As bird host cells, there may be mentioned, for example, fibroblasts,pluripotent stem cells, primordial germ cells and the like. Arecombinant bird host which is constructed by infecting a bird host withsaid lentivirus vector, and a method of constructing said recombinantbird host also constitute aspects of the present invention.

The lentivirus vector to be used in the practice of the invention ispreferably a heterologous gene-containing one.

The heterologous gene to be introduced into birds using the lentivirusvector in accordance with the invention is not particularly restrictedbut is constituted of a marker gene or a structural gene for theexpression of a desired protein, a promoter gene controlling theexpression of those genes, a secretory signal gene and the like. Theheterologous gene preferably contains a structure gene and, morepreferably, further contains a marker gene.

As the marker gene, there may be mentioned the neomycin resistance gene,LacZ (β-galactosidase gene), and the gene coding for a fluorescentprotein such as GFP (green fluorescent protein). The lentivirus vectorto be used in the practice of the invention is preferably one with theGFP gene and/or LacZ inserted therein.

The structural gene for the expression of a desired protein is notparticularly restricted but includes, for example, genes coding forantibodies useful in the genetic industry, such as human monoclonalantibodies, enzymes or the like, or genes coding for single-strandantibodies. Furthermore, genes coding for other useful physiologicallyactive substances can also be used. Particularly preferred arestructural genes coding for exogenous antibodies, for example genescoding for antibodies having a constant region of the human IgG classand genes coding for antibodies having a constant region of the humanIgG1, quail IgG2, chicken IgG2 or mouse IgG2 subclass, since suchantibodies are abundantly accumulated in eggs.

As preferred structural genes for the expression of the desired proteinsmentioned above, there may be mentioned structural genes coding forchimera antibodies. The term “chimera antibody” refers to an antibodycomprising two or more different inherited characters.

The antibodies for medical use so far produced by mouse hybridomas areof the mouse origin and, therefore, it is a problem that whenadministered into the human body, they cause rejection by the immunesystem. As the chimera antibodies, there may be mentioned, for example,those chimera antibodies in which the above drawback has been remediesand which cause no rejection, for example anti-human CD2 antibodies,anti-CD20 receptor antibodies, anti-TNF antibodies and the like. Some ofthem are already on the market as medicines.

By using such human monoclonal antibody genes or chimera antibody genesas the genes to be introduced into birds to give the G0 transgenicchimeric birds according to the invention, it becomes possible toproduce antibodies for medical use, which have so far been difficult toproduce, in large amounts and at low cost.

As the promoter gene, there may be mentioned constitutive promoters.When an antibody gene is controlled under a constitutive promoter, theexpression of the antibody gene is favorably stable. As a more preferredconstitutive promoter, there may be mentioned the chicken β actinpromoter.

The G0 transgenic chimeric birds and offspring thereof as constructed inaccordance with the invention by introduction of an exogenous antibodygene by means of a lentivirus vector can produce the transgene-derivedprotein in blood, egg white or yolk. The proteins thus produced undergosugar chain modification in bird cells and this is advantageous from theactivity and stability viewpoint.

The birds to be used in the practice of the invention are notparticularly restricted but there may be mentioned, for example,chickens, turkeys, ducks, ostriches, quails and other domestic fowlsdomesticated for eating as meats or collecting eggs as well as petbirds. Among them, chickens and quails are readily available andpreferred because of their being prolific as egg-laying species. Morepreferred are chickens.

The method of constructing G0 transgenic chimeric birds and offspringthereof according to the invention is now described.

The method of constructing G0 transgenic chimeric birds and offspringthereof according to the invention comprises infecting fertilized birdegg early embryos with a lentivirus vector comprising a coat proteincontaining VSV-G, and incubating the embryos.

As an example of such method for construction, there may be mentionedthe method comprising infecting early embryos at the blastodermic stage(stageX) just after laying of fertilized bird eggs with areplication-defective lentivirus vector, and incubating the embryos.Also employable is the method which comprises incubating fertilized birdeggs, infecting early embryos thereof at the state succeeding theblastodermic stage just after being laid with a replication-defectivelentivirus vector, and incubating the embryos. As another example of themethod of the invention, there may be mentioned the method comprisingincubating fertilized bird eggs, infecting early embryos thereof afterat least 24 hours from the start of incubation with areplication-defective lentivirus vector, and incubating the embryos.

More preferred is the method comprising microinjecting areplication-defective lentivirus vector into the heart or blood vesselformed in the early embryos.

In carrying out the method of constructing G0 transgenic chimeric birdsand offspring thereof according to the invention, areplication-defective lentivirus vector is preferably microinjected intofertilized eggs just after being laid (at stage X).

As for the early development of a fertilized egg after being laid in achicken, for example, the egg fertilized in the oviduct begins cleavagein about 1.5 hours after fertilization. The egg that has begun discoidalcleavage with the cytoplasm still undivided cleaves over a period of 1day until discharge from the body, to form an embryo, called blastoderm,consisting of about 60 thousand cells (blastodermic stage). Thisblastoderm is observed as a white ring, 3 to 4 mm in diameter, in thecentral part of the yolk. This embryo cleaves into an upper and a lowerlayer to form a blastocoel. The egg is laid when the hypoblast isformed, the primitive streak is then formed, and the blastoderm takes athree-layer structure consisting of an upper layer, a middle layer and alower layer. Three germ layers are thus formed. Then, afterembryogenesis and growth, hatching occurs on the 22nd day afterovulation. The blastodermic stage is also called stage X, andreproductive cells are formed from a part of the cells at this stage.Therefore, fertilized eggs at this stage are conventionally used astargets of gene transfer.

In the practice of the invention, the stage for introducing aheterologous gene into embryos is not particularly restricted but may bebefore stage X, at stage X or after stage X. From the viewpoint thatsilencing hardly occurs, the heterologous gene is preferably introducedinto embryos after stage X, more preferably after the lapse of 48 hoursfrom the start of incubation, most preferably at 55 hours from the startof incubation.

In the practice of the invention, the time when fertilized eggs at theblastodermic stage just after being laid were placed under environmentalconditions suited for hatching (in the case of chickens, for example, atemperature of 37.7 to 37.8° C. and a humidity of about 50 to 70%) wastaken as time 0 (zero), and microinjection was carried out at timedintervals. The formation of a blood circulatory system on the yolk wasobserved and the pulsation of an organ to differentiate into the heartcould be observed after 36 hours from the start of incubation in quails,or after about 50 hours from the start of incubation in chickens.

For the hatching of fertilized eggs after the above gene transfer, themethod using artificial eggshells as developed by the present inventors(Kamihira, M. et al. (1998) Develop. Growth Differ., 40, 449), forexample, can be applied.

As the lentivirus vector, the transgene and the bird to be subjected togene transfer, which are to be used in carrying out the method forconstruction of the invention, there may be mentioned the same ones asdescribed hereinabove.

In the method for construction of the invention, the “gene not derivedfrom lentivirus” includes marker genes, structural genes, promoter genesand secretory signal genes.

In carrying out the method for construction of the invention, the timefor lentivirus vector infection is preferably at the blastodermic stage.The time for lentivirus vector infection is also preferably at a periodsucceeding the blastodermic stage and, in that case, the site oflentivirus vector infection is preferably in the early heart or earlyblood vessel.

In carrying out the method for construction of the invention, the methodof lentivirus vector infection is preferably the microinjection method.

In carrying out the method for construction of the invention, injection,preferably microinjection, of a lentivirus vector having a titer of notlower than 1×10⁵ cfu/mL, more preferably not lower than 1×10⁶ cfu/mL, ispreferred, since, then, the gene transfer can be carried outefficiently.

In the practice of the invention, the virus titer is measured by theX-Gal staining method or fluorescent protein detection method.

The birds constructed by the gene transfer into fertilized eggs thereofby the method for construction of the invention grow as transgenic birdscarrying the transgene in a mosaic manner in their somatic cells. Thesefirst generation transgenic birds are referred to as G0 transgenicchimeric birds.

The G0 transgenic chimeric birds and offspring thereof obtained by suchmethod for construction of the invention also constitute an aspect ofthe present invention.

The offspring of the G0 transgenic chimeric birds of the invention canbe produced by any of the breeding technologies known in the art. Therelated technologies are well known in the relevant field of art andinclude, but are not limited to, hybridization, inbreeding, backcross,multiple cross, interspecific hybridization and the like.

More specifically, each individual of the second generation and thirdgeneration born as a result of mating of a G0 transgenic chimeric birdwith a non-transgenic bird or mating of a G0 transgenic chimeric birdwith a G0 transgenic chimeric bird, when it has developed from areproductive cell containing the transgene on a chromosome thereof,grows as an individual containing the transgene in the somatic cells ofthe whole body. The offspring inheriting the transgene from G0transgenic chimeric bird individuals are referred to successively as G1,G2, G3 . . . transgenic birds.

By mating a G0 transgenic chimeric bird according to the invention witha non-transgenic bird of the same species or with a mating type G0transgenic chimeric bird, it becomes possible to transmit the transgeneto their offspring and at the same time to construct fully transgenicbirds carrying the transgene in the somatic cells of the whole body. Ascompared with G0 transgenic chimeric birds, such fully transgenic birdscan be expected to produce the transgene-derived recombinant protein inincreased amounts, since the proportion of the transgene-containingsomatic cells is higher. Furthermore, by establishing a line oftransgenic birds capable of stably transmitting the transgene, itbecomes possible to stabilize the quality as a protein productionsystem.

In accordance with the invention, G0 transgenic chimeric birds can beconstructed by hatching fertilized bird egg early embryos infected witha lentivirus vector comprising a coat protein pseudotyped with VSV-G.The method of constructing G0 transgenic chimeric birds and offspringthereof according to the invention makes it possible to introduce thegene coding for a physiologically active protein, for example anantibody for medical use, to construct birds capable of efficientlyexpressing the antibody in the blood or eggs. Furthermore, the method ofproducing proteins using the G0 transgenic chimeric birds and offspringthereof according to the invention enables efficient antibody productionby constructing G0 transgenic chimeric birds and offspring thereofcapable of producing an antibody for medical use, for example, andrecovering and purifying the antibody from the serum and/or eggs of thebirds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the vector construct pLenti6/CMV-lacZ forβ-galactosidase expression prepared in Example 1 to be described below.Blasticidin denotes the blasticidin resistance gene, and Ampicillindenotes the ampicillin resistance gene. Ψ denotes the packaging signalsequence. CMVPro denotes the CMV promoter gene. LacZ denotes the genefor β-galactosidase expression. 5′LTR and 3′LTRΔU3 respectively denotethe CMV long terminal repeat sequences.

FIG. 2 shows the differences in β-galactosidase activity among cellspecies. The abscissa denotes the amount added of the virus solutionhaving a titer of (1.7±0.3)×10⁶ cfu/mL. The ordinate denotes theβ-galactosidase activity expressed in terms of units. Mock denotes acontrol.

FIG. 3 shows the structure of the vector construct pLenti/cDf-ΔAsPE-Wfor anti-prion scFvFc and green fluorescent protein (GFP) expression.bla denotes the blasticidin resistance gene. Ψ denotes the packagingsignal sequence. pact denotes the chicken β-actin promoter gene. scFv-Fcdenotes the single strand antibody structural gene. EGFP denotes thegreen fluorescent protein (GFP) structural gene.

BEST MODES FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in detail. Theseexamples are, however, by no means limitative of the scope of theinvention.

EXAMPLE 1 Preparation of Vector Construct pLenti6/CMV-lacZ forβ-galactosidase Expression

The pLenti6/CMV-lacZ vector construct was constructed in the followingmanner.

First, pLenti6/TOPO-GFP was constructed in the following manner. Twoprimers for amplifying GFP, namely 5′-CACCATGAGCAAGGGC-3′ (SEQ ID NO:1)and 5′-TCACTTGTACAGCTCGTCCA-3′ (SEQ ID NO:2), were chemicallysynthesized. Using pGreen Lantern (product of Gibco) as a template, PCRwas carried out to amplify a GFP fragment. This amplification productsolution was subcloned into the pLenti6/V5-D-TOPO vector according tothe manual attached to pLenti6/V5 Directional TOPO Cloning Kit (productof Invitrogen Corporation), whereby pLenti6/TOPO-GFP was constructed.

Furthermore, pLNΔAβ was constructed in the following manner.

-   1. A Rous sarcoma virus (RSV) promoter fragment was cleaved from the    plasmidpLXRN (product of Clontech Laboratories, Inc.) with the    restriction enzymes XhoI and HindIII and inserted into the plasmid    pBluescriptIISK(+) (product of Stratagene) at the XhoI-HindIII site.    A plasmid, pBlue/RSV, was thus constructed.-   2. A β-galactosidase (β-Gal) gene fragment was cleaved from the    plasmid pCMVβ (product of Clontech Laboratories, Inc.) with the    restriction enzyme NotI and inserted into the plasmid pZeoSV2 (+)    (product of Invitrogen Corporation) at the NotI site. The plasmid    having a structure resulting from insertion of the β-Gal gene in the    same direction as the T7 promoter was designated as pZeo/lacZ.-   3. A RSV promoter fragment was cleaved from pBlue/RSV with the    restriction enzymes XhoI and PstI. A β-Gal gene fragment was cleaved    from pZeo/lacZ with the restriction enzymes PstI and XhoI. The above    two fragments cleaved were joined to a vector fragment prepared by    treatment of the plasmid pLNHX (product of Clontech Laboratories,    Inc.) with the restriction enzyme XhoI, whereby a plasmid, pLNRβ,    was constructed.-   4. A fragment containing the series of the Moloney murine sarcoma    virus (MoMuSV) 5′-long terminal repeat (LTR), virus packaging signal    and neomycin resistance (Neor) gene was cleaved from pLNHX with the    restriction enzymes SacII and XhoI and joined to a vector fragment    prepared by treatment of pLXRN with the restriction enzymes SacII    and XhoI. A plasmid, pLXL, was thus constructed.-   5. A β-Gal gene fragment was cleaved from pZeo/lacZ with the    restriction enzymes HindIII and XhoI and joined to a vector fragment    prepared by treatment of pLXL with the restriction enzymes HindIII    and XhoI. A plasmid, pLZL, was thus constructed.-   6. A 5′ region fragment of the hybrid promoter (Miw promoter)    derived from the RSV promoter and chicken β-actin (Act) promoter was    amplified from the plasmid pMiwZ (Suemori et al., 1990, Cell Diff.    Dev. 29:181-185) byPCR (94° C./15 seconds→55° C./30 seconds→72° C./1    minute and 30 seconds: 35 cycles; KOD-Plus-DNA polymerase (product    of TOYOBO CO., LTD.)) using, as primers, two chemically synthesized    oligonucleotides, 5′-cggtctagaggaattcagtggttcg-3′ (SEQ ID NO:3) and    5′-ccaggatccgacgttgtaaaacgacg-3′ (SEQ ID NO:4; the underlined    portion being the BamHI restriction site), followed by cleavage    thereof with the restriction enzymes BamHI and MunI. The cleaved    fragment was inserted into the plasmid pGREEN LANTERN-1 (product of    Gibco BRL) at the BamHI-MunI site. A plasmid, pGmiw5′, was thus    constructed.-   7. AMiw promoter 5′ side central region fragment was cleaved from    pMiwZ with the restriction enzymes MunI and ClaI. By inserting that    fragment into pGmiw5′ at the MunI-ClaI site, a plasmid, pGmiw5′-2,    was constructed.-   8. A fragment containing the Miw promoter 5′ region and 5′ side    central region was cleaved from pGmiw5′-2 with the restriction    enzymes BamHI and EcoRI. By inserting that fragment into    pBluescriptIISK(+) at the BamHI-EcoRI site, a plasmid, pBlue/Miw5′,    was constructed.-   9. AMiw promoter 3′ region fragment was amplified frompMiwZ by PCR    (98° C./15 seconds→60° C./30 seconds→72° C./30 seconds: 35 cycles)    using, as primers, two chemically synthesized oligonucleotides,    5′-ccaaagcttgccgcagccattgcctttt-3′ (SEQ ID NO:5; the underlined    being the HindIII restriction site) and    5′-atacctaggggctggctgcggaggaac-3′ (SEQ ID NO:6; the underlined    portion being the BlnI restriction site), followed by cleavage    thereof with the restriction enzymes HindIII and BlnI. By joining    the cleaved fragment to a vector fragment prepared by treatment of    pLXL with the restriction enzymes HindIII and BlnI, a plasmid,    pLMiw3′, was constructed.-   10. AMiw promoter 3′ side central region fragment was cleaved from    pMiwZ with the restriction enzymes EcoRI and MboII. Furthermore, a    Miw promoter 3′ region fragment was cleaved from pLMiw3′ with the    restriction enzymes MboII and KpnI. The two fragments cleaved were    inserted into pBlue/Miw5′ at the EcoRI-KpnI site. A plasmid,    pBlue/Miw, was thus constructed.-   11. A fragment containing the full-length Miw promoter was cleaved    from pBlue/Miw with the restriction enzymes BamHI and BlnI. This    fragment was joined to a vector fragment prepared by treatment of    pLXL with the restriction enzymes BamHI and BlnI. A plasmid, pLML,    was thus constructed.-   12. An Act promoter fragment was cleaved from pLML with the    restriction enzymes SmaI and XbaI. The fragment was inserted into    pBluescriptIISK(+) at the EcoRV-XbaI site. A plasmid, pBlue/Act, was    thus constructed.-   13. A Miw promoter fragment was cleaved from pLML with the    restriction enzymes HindIII and BglII. This fragment was joined to a    vector fragment prepared by treatment of pLZL with the restriction    enzymes HindIII and BamHI. A plasmid, pLMβL, was thus constructed.-   14. An Act promoter fragment was cleaved from pBlue/Act with the    restriction enzymes SalI and BlnI. A β-Gal gene fragment was cleaved    from pLMβL with the restriction enzymes BlnI and BglII. The    above-mentioned two fragments cleaved were joined to a vector    fragment prepared by treatment of pLNRβ with the restriction enzymes    XhoI and BglII. Aplasmid, pLNAβ, was thus constructed.-   15. An intron-deficient actin (ΔAct) promoter fragment was amplified    from pMiwZ by PCR (98° C./15 seconds→60° C./30 seconds→68° C./2    minutes: 30 cycles) using, as primers, two chemically synthesized    oligonucleotides, 5′-tttagctagctgcagctcagtgcatgcac-3′ (SEQ ID NO:7;    the underlined portion being the NheI restriction site) and    5′-ataatctagaaacgcagcgactcccgc-3′ (SEQ ID NO:8; the underlined    portion being the XbaI restriction site), followed by cleavage of a    fragment containing a part of the ΔAct promoter with the restriction    enzymes XhoI (the XhoI restriction enzyme site occurring in the    fragment amplified) and XbaI. A fragment containing the remaining    portion of the ΔAct promoter and the β-Gal gene was cleaved from    pLNAβ with the restriction enzymes BlnI and BglII. The above two    fragments cleaved were joined to a vector fragment prepared by    treatment of pLNAβ with the restrictionenzymesXbaIandBglII.    Aplasmid, pLNΔAβ, was thus constructed.

The lacZ gene prepared by cleaving pLNΔAβ with SpeI and XhoI was joinedto pLenti6/TOPO-GFP at the SpeI-XhoI site. pLenti6/CMV-lacZ was thusconstructed.

The structure of pLenti6/CMV-lacZ is shown in FIG. 1.

EXAMPLE 2 Preparation of Replication-Defective Lentivirus Vector forβ-galactosidase Expression

For preparing a lentivirus vector, 293FT packaging cells (product ofInvitrogen Corporation) (5×10⁶ cells) were sown and cultured in culturedishes with a diameter of 100 mm. On the next day, at 90% confluence,the medium was exchanged for antibiotic-free fresh DMEM (Dulbecco'smodified Eagle medium), and the defective vector constituent elementswere introduced in the form of four plasmids (independently preparedfrom ViraPower Packaging Mix; 6.5 μg of gag/pol-encoding pLP1, 2.5 μg ofRev-encoding pLP2, 3.5 μg of pLP/VSV-G encoding coat protein VSV-G and10 μg of pLenti6/CMV-lacZ prepared in Example 1, per culture dish) bythe lipofection technique. On the day after gene transfer, mediumexchange was made and, at 48 hours after transfection, the virusparticle-containing culture supernatant was recovered and deprived ofimpurities by filtration through a 0.45-μm cellulose acetate filter(product of Advantec). This culture supernatant was centrifuged at25,000 rpm at 4° C. for 1.5 hours to cause precipitation of the virus.The supernatant was removed, 50 μl of TNE (50 mMTris-HCl (pH 7.8), 130mM NaCl, 1 mM EDTA) was added to the virus particle-containingprecipitate and, after overnight standing at 4° C. and after thoroughsuspending, the virus solution was recovered. Polybrene (product ofSigma) was added to the solution obtained to a concentration of 8 μg/mL,and the resulting mixture was used as the virus solution.

The thus-obtained high titer virus vector had a titer of 3.7×10⁶ cfu/mL.

The virus titer measurement was carried out as follows. On the daybefore the measurement, HeLa cells (human-derived epithelial cell line,obtained from the American Type Culture Collection) were sown andcultured on 24-well dishes (1.5×10⁴ cells/dish). To each dish was added1 mL of a 10² to 10⁶-fold dilution of the virus solution and, after 48hours, the cells were washed with two portions of PBS(phosphate-buffered physiological saline). Then, a fixing solution(200-fold dilution of 50% glutaraldehyde (product of Tokyo Kasei KogyoCo., Ltd.) with PBS) was added in an amount sufficient for the cells tobe immersed, followed by 10 minutes of standing at 4° C. Thereafter, thefixing solution was discarded, the cells were washed with three portionsof PBS, an X-Gal staining solution (5 mM of K₃Fe(CN) 6, 5 mM ofK₄Fe(CN)₆, 2 mM of MgCl₂, 1 mg/mL of5-bromo-4-chloro-3-indolyl-β-D-galactoside (product of Takara Shuzo Co.,Ltd.) was then added and, after at least 3 hours of staining at 37° C.,the proportion of the cells stained blue was determined. The titer wascalculated as follows:Virus titer=(number of cells)×(dilution factor)×(expression ratio)(cfu/mL)

EXAMPLE 3 Preparation of Chicken and Quail Fibroblasts

Chicken fibroblasts (CEF) and quail fibroblasts (QEF) were prepared asfollows.

Fertilized chicken or quail eggs were incubated in an incubator (ShowaFuranki model P-008) for 5 days (37.9° C., 65% humidity). Embryos weretaken out of eggshells, transferred to culture dishes and washed with0.1 M sterile phosphate buffer (pH 7.4), and the head, wings, limbs andinternal organs were removed using scissors. Each embryo tissue wastransferred to a centrifuge tube, 2 mL of a 0.25% trypsin solution(product of Sigma) was added, and cells were disrupted and suspended byrepeating pipetting. The suspension was incubated at 37° C. for 10minutes, the reaction was then stopped by addition of Dulbecco'smodified Eagle medium (product of Invitrogen Corporation), and cellswere separated by 5 minutes of centrifugation at 1000 rpm. Thesupernatant was discarded, and Dulbecco's modified Eagle medium wasagain added, and the cells were sown on a culture dish. The cultivationwas carried out in a 5% CO₂ atmosphere at 37° C.

EXAMPLE 4 Differences in Lentivirus Vector Titer among Cell Species

The lentivirus vector prepared in Example 2 was introduced into culturedcells and the titer of the virus particles expressed was determined. Inthis way, the differences in the infectivity of the lentivirus vectoramong cell species were examined.

The cells used were HeLa (obtained from the American Type CultureCollection), NIH3T3 (NIHswiss mouse-derived fibroblastoid cell line,obtained from the American Type Culture Collection), and CEF and QEFcells prepared in Example 3, and 1.5×10⁴ cells of each species were sown(Dulbecco's modified Eagle medium). On the next day, the medium wasexchanged for a virus-containing medium and, after 2 days of incubation,the virus titer in the supernatant was assayed by the method describedin Example 2.

In the same manner, each cell species was infected with the lentivirusfor β-galactosidase expression constructed in Example 2, and theactivity of the β-galactosidase expressed was measured and used as anindication of the infectivity of the lentivirus in each cell species.

The β-galactosidase activity measurement was carried out in thefollowing manner.

Cultured cells were treated with a 0.25% trypsin solution (product ofSigma), and cells were collected by 10 minutes of centrifugation at 1500rpm. Each pellet was washed with 0.1 M phosphate buffer (pH 7.5) and,after addition of 0.8 mL of a reaction buffer (10 mM of KCl, 1 MM ofMgCl₂, 0.1% of Triton X-100 (product of Wako Pure Chemical Industries),5 mM of 2-mercaptoethanol (product of Wako Pure Chemical Industries), 2mM of phosphate buffer (pH 7.5)), cells were disrupted by sonication togive a cell solution.

A 0.6-mL portion of each cell solution was incubated at 37° C. for 8hours and, then, 0.1 mL of a preliminarily warmed solution of 4 mg/mLo-nitrophenyl β-D-galactopyranoside (ONPG) (product of Sigma) in 0.1Mphosphate buffer (pH 7.5) was added. After allowing the reaction toproceed, 0.3 mL of 1 M Na₂CO₃ (product of Wako Pure Chemical Industries)was added, and the absorption intensity at the wavelength of 420 nm wasmeasured using an absorptiometer.

The β-galactosidase activity was expressed in terms of ONPG units (1unit: activity corresponding to the formation of 1 μmol of o-nitrophenolper minute). Three runs were carried out, and the mean thereof wasreported as the β-galactosidase activity.

Each cell species was infected with the lentivirus vector forβ-galactosidase expression and the titer of the virus particles formedwas determined. In this way, comparisons were made among the cellspecies with respect to the infectivity of the lentivirus.

The results are shown in Table 1. In Table 1, “Relative titer” refers tothe percent titer in each cell species with the virus titer on theoccasion of infection in HeLa being taken as 100. TABLE 1 Hela NIH3T3CEF QEF (Human) (Mouse) (Chicken) (Quail) Virus titer 8.5 ± 2.1 × 10⁵3.5 ± 0.5 × 10⁴ 2.5 ± 0.5 × 10⁵ 2.2 ± [cfu/mL] 0.5 × 10⁴ Relative 1004.1 29.4 2.6 titer(%)

Examination of the relative infectivity levels in comparison withhuman-derived HeLa cells in which high infectivity of lentiviruses hasbeen confirmed revealed that the lentivirus comprising a coat proteinpseudotyped with VSV-G shows an infectivity of about 30% inchicken-derived fibroblasts as compared with the infectivity in HeLa.

Each cell species was infected with the lentivirus vector forβ-galactosidase expression at various virus amount levels, and theenzyme activities due to the transgene were examined. Such enzymeactivities serve as indicators of the infectivity of the lentivirus andof the gene transfer capacity. The results are shown in FIG. 2.

The VSV-G pseudotyped lentivirus vector caused the chicken fibroblaststo show a β-galactosidase activity about half that in HeLa.

Whereas there is no report about the infection of bird cells withlentivirus vectors, it was confirmed here that gene transfer can beaccomplished with an infectivity of half to one third as compared withHeLa by pseudotyping the coat protein with VSV-G.

EXAMPLE 5 Preparation of Vector Construct pLenti/cDf-ΔAsPE-W foranti-prion scFvFc and green fluorescent protein (GFP) expression

The vector construct pLenti/cDf-ΔAsPE-W for anti-prion scFvFc and GFPexpression was constructed in the following manner. The scFvFc region ofthe construct pMSCV/GΔAscFv-Fc previously constructed by the presentinventors (WO 2004/016081) was cleaved from that construct by cleavagewith NheI and inserted into pBluescriptKS (−) (product of Stratagene) atthe XbaI site to construct pBlue-scFvFc. The scFvFc region was cleavedfrom this pBlue-scFvFc by cleavage with XbaI and NotI, and the actinpromoter region was cleaved from pMSCV/GΔAscFv-Fc with NotI and NheI.These two fragments were simultaneously inserted into pBluescriptKS (−)at the NotI site to construct pBlue-Pact-scFvFc. The GFP region of theabove-mentioned pLenti6/TOPO-GFP was cleaved therefrom with SpeI andXbaI and inserted into pBluescriptKS(−) at the SpeI-XbaI site toconstruct pBlueGFP. The PGK promoter region was cleaved from pMSCVpuro(product of Clontech Laboratories, Inc.) with XhoI and PstI and insertedinto pBlueGFP at the XhoI-PstI site to construct pBlue-Ppgk-GFP. The PGKpromoter region and GFP were cleaved from this pBlue-Ppgk-GFP with XhoIand again inserted into pBluescriptKS(−) at the XhoI site to constructpBlueXPpg-GFP. The PGK promoter region and GFP were cleaved frompBlueXPpg-GFP with ClaI and KpnI and inserted into pLenti6/TOPO-GFP atthe ClaI-KpnI site to construct pLenti-PpgkGFP. For replacing GFP withenhanced GFP (EGFP), EGFP was amplifiedby PCR using pIRES2-EGFP (productof Clontech Laboratories, Inc.) as a template as well as the primerEGFPdirect(BamHI) 5′-ATTGGATCCACACGATGATAATATGGCCAC-3′ (SEQ ID NO:9; theunderlined portion being the BamHI site) and the primerEGFPreverse(KpnI) 5′-ATTGGTACCAGGCCGCTTTACTTGTACAG-3′ (SEQ ID NO: 10;the underlined portion being the KpnI site). The amplified PCR productwas cleaved with BamHI and KpnI and inserted into pLenti-PpgkGFP at theBamHI-KpnI site to construct pLenti-PpgkEGFP. The actin promoter andscFvFc regions were cleaved from pBlue-Pact-scFvFc with ClaI andinserted into pLenti-PpgkEGFP at the ClaI site to constructpLenti-PactscFvFc-PpgkEGFP. For insertion of a central DNA flap (cDf),the cDf was amplified by PCR using pLP1 (product of InvitrogenCorporation) as a template as well as the primer cDfdirect5′-ACGTCTAGAAGACAGCAGTACAAATGGCAGTATT-3′ (SEQ ID NO:11; the underlinedportion being the XbaI site) and the primer cDfreverse5′-AAGTCTAGACCAAACTGGATCTCTGCTGTCC-3′ (SEQ ID NO:12; the underlinedportion being the XbaI site). The amplified PCR product was cleaved withXbaI, and pLenti/cDf-ΔAsPE was constructed. Finally, the WoodchuckHepatitis Virus Posttranscriptional regulatory element (WPRE) wasamplified by PCR using the primer WPRE(direct KpnI)5′-ACCGGTACCAATCAACCTCTGGATTACAAA-3′ (SEQ ID NO:13; the underlinedportion being the KpnI site) and the primer WPRE(reverse KpnI)5′-ATAGGTACCCAGGCGGGGAGGC-3′ (SEQ ID NO:14; the underlined portion beingthe KpnI site). This amplified product was cleaved with KpnI andinserted into pLenti/cDf-ΔAsPE at the KpnI site to constructpLenti/cDf-ΔAsPE-W (FIG. 3).

EXAMPLE 6 Construction of G0 Transgenic Chimeric Chickens for theExpression of scFvFc and GFP

First, the virus vector was prepared by the method described in Example2. The titer measurement of the thus-prepared virus vector containingGFP as a marker was carried out in the following manner. On the daybefore the measurement, HeLa cells were sown and cultured on 24-welldishes (1.5×10⁴ cells/dish). To each dish was added 1 mL of a 10² to10⁶-fold dilution of the virus solution and, after 48 hours, the mediumwas exchanged for PBS, and the proportion of the cells manifesting theexpression of the GFP gene was determined under a fluorescentmicroscope. The titer was calculated as follows:Virus titer=(number of cells)×(dilution factor)×(expression ratio)(cfu/mL)

The titer of the thus-prepared virus was 2.2×10⁷ cfu/ml to 7.7×10⁹cfu/ml.

This virus solution was introduced into chicken embryos just afterlaying by the method described in Japanese Kokai Publication2002-176880. The chicken embryos after virus vector introduction wereincubated for hatching by the method described in Japanese KokaiPublication 2002-176880. G0 transgenic chimeric chickens for scFvFc andGFP gene expression were thus constructed.

EXAMPLE 7 Serum scFvFc Concentration Measurement by ELISA

The G0 transgenic chimeric chickens constructed in Example 6 were raisedfor 20 to 40 days for growing chicks and, then, blood samples wereobtained by blood drawing from the subalary vein. Each blood sample wasincubated at 37° C. for 1 hour and then centrifuges at 15, 000 rpm for 5minutes, and the supernatant was used as a serum sample. Anti-Fcantibody (product of Cosmo Bio co., ltd.) diluted with PBS was placed ineach well of an ELISA plate (100 pg/well), followed by overnightstanding at 4° C. Each well was washed with three 200-μl portions of a0.05% solution of Tween 20 in PBS and, then, 150 μl of PBS-0.05% Tween20 solution-2% skim milk was added to each well. After 2 hours ofstanding at room temperature, each well was washed with three 200-μlportions of a 0.05% solution of Tween 20 in PBS, then 100 μl of eachserum sample collected was added thereto, and the plate was allowed tostand at room temperature for 2 hours. Thereafter, each well was washedwith three 200-μl portions of a 0.05% solution of Tween 20 in PBS, and100 μl of peroxidase (POD)-labeled anti-human Fc antibody (product ofCosmoBio co., ltd.) dilutedwith a 0.05% solution of Tween 20 in PBS wasadded to each well, and the plate was allowed to stand at roomtemperature for 1 hour. The wells were washed with four portions of a0.05% solution of Tween 20 in PBS, and 100 ul of a color former solution(prepared by adding 10 μl of an aqueous solution of hydrogen peroxide(product of Wako Pure Chemical Industries) to a solution of 10 mg ofo-phenylenediamine (product of KATAYAMA CHEMICAL INDUSTRIES Co., Ltd.)in 1 ml of methanol (diluted by distilled water to 100 ml)) was added toeach well. After the lapse of 30 minutes to 1 hour, the reaction wasstopped by adding 50 μl of 8 M concentrated sulfuric acid to each well,the fluorescence intensity at 490 nm was measured using a plate reader,and the concentration was calculated using a standard calibration curve.This standard calibration curve was constructed using standard Fc(product of Cosmo Bio co., ltd.).

The thus-measured serum scFvFc concentrations are shown in Table 2.TABLE 2 Individual's Concentration No. (ng/ml) B10 23.4 B13 18.2 B16 7.6B21 7.6 B23 20.8 B24 15.5 B25 10.3 B28 23.4 B30 15.5 B31 5

In this way, by carrying out gene transfer in fertilized chicken eggearly embryos using a lentivirus vector comprising a coat proteinpseudotyped with VSV-G, it becomes possible to construct G0 transgenicchimeric chickens capable of expressing the desired protein.

INDUSTRIAL APPLICABILITY

By incubating, for hatching, fertilized bird egg early embryos infectedwith a lentivirus vector comprising a coat protein pseudotyped withVSV-G according to the invention, it is possible to construct G0transgenic chimeric birds.

Furthermore, the method of constructing G0 transgenic chimeric birds andoffspring thereof makes it possible to construct birds capable ofefficiently expressing a physiologically active protein, for example anantibody for medical use, in the blood and/or eggs as a result ofintroduction of the gene coding for such protein or antibody.Furthermore, the method of producing protein which uses the G0transgenic chimeric birds and offspring thereof of the invention makesit possible to produce an antibody for medical use, for example, withgood efficiency by constructing G0 transgenic chimeric birds andoffspring thereof capable of producing the antibody and recovering andpurifying the antibody from the serum and/or eggs of the birds.

1. A lentivirus vector which comprises a coat protein containing VSV-G.2. The vector according to claim 1 which is replication-defective. 3.The vector according to claim 1 which further contains a heterologousgene.
 4. The vector according to claim 3 wherein the heterologous genecontains a structural gene.
 5. The vector according to claim 4 whereinthe heterologous gene further contains a marker gene.
 6. A recombinantbird host which is constructed by infecting a bird host with the vectoraccording to claim
 1. 7. The recombinant bird host according to claim 6wherein the vector contains a structural gene.
 8. The recombinant birdhost according to claim 6 wherein the bird host is a fertilized eggearly embryo.
 9. A transgenic chimeric bird which is constructed byinfection with the vector according to claim 1, and offspring thereof.10. The transgenic chimeric bird and offspring thereof according toclaim 9 wherein the vector contains a structural gene.
 11. A method ofconstructing a recombinant bird host which comprises infecting a birdhost with the vector according to claim
 1. 12. The method according toclaim 11 wherein the bird host is a fertilized egg early embryo.
 13. Amethod of constructing a G0 transgenic chimeric bird and offspringthereof which comprises infecting fertilized bird egg early embryos witha lentivirus vector comprising a coat protein containing VSV-G, andincubating the embryos.
 14. The method of constructing a G0 transgenicchimeric bird and offspring thereof according to claim 13 wherein thelentivirus vector is replication-defective.
 15. The method ofconstructing a G0 transgenic chimeric bird and offspring thereofaccording to claim 13 wherein the lentivirus vector is an HIV-derivedone.
 16. The method of constructing a G0 transgenic chimeric bird andoffspring thereof according to claim 13 wherein the lentivirus vector isone with the GFP gene and/or LacZ integrated therein.
 17. The method ofconstructing a G0 transgenic chimeric bird and offspring thereofaccording to claim 13 wherein the time for lentivirus vector infectionis at a period succeeding the blastodermic stage.
 18. The method ofconstructing a G0 transgenic chimeric bird and offspring thereofaccording to claim 17 wherein the site of lentivirus vector infection isin the heart or blood vessel formed in the early embryos.
 19. The methodof constructing a G0 transgenic chimeric bird and offspring thereofaccording to claim 13 wherein the method of lentivirus vector infectionis the microinjection method.
 20. The method of constructing a G0transgenic chimeric bird and offspring thereof according to claim 13wherein the bird is a chicken.
 21. The method of constructing a G0transgenic chimeric bird and offspring thereof according to claim 13wherein a lentivirus vector having a titer of not lower than 1×10⁵cfu/mL is injected.
 22. A G0 transgenic chimeric bird and offspringthereof which is constructed by the method according to claim 13.